[Analysis of molecular genetics and clinical characteristics of 3 children with Waardenburg syndrome].

Objective:To analyze the molecular genetics and clinical characteristics of 3 children with syndromic deafness were analyzed to clarify their causative genes and genetic characteristics. Methods:The medical records of 3 children and their parents were collected and analyzed, including physical examination, hearing evaluation, temporal bone CT, and cranial MRI. Whole-exome sequencing（WES） was used to screen for pathogenic gene variants, and Sanger sequencing was used to verify the candidate positive variants in the probands and their parents. Results:All 3 patients were female with normal intelligence. Patient 1 and 3 had a family history of deafness, which conformed to the pattern of autosomal dominant inheritance. All three patients had bilateral profound sensorineural hearing impairment with bright-blue sclera. Other phenotypes included hypertelorism（patient 1）, multiple dyschromatosis（patient 2）, and yellowish hair（patient 2）, blepharoptosis（patient 3）. Patient 3 had bilateral vestibular enlargement, internal auditory canal enlargement, and bilateral inner ear malformations. Mother of patient 1 had only left mild hearing impairment; mother of patient 3 had bilateral hearing impairment with unilateral bright-blue sclera and yellowish hair. WES detected heterozygous variants, PAX3 c.811C>T, MITF c.632T>C, and SOX10 c.1359_1360 insGCCCCACA, in patient 1, 2, and 3, respectively. The variants in patient 1 and 3 were inherited from their mothers who had hearing impairment, and MITFvariant in patient 2 may be a spontaneous variation. The final diagnoses were that patient 1 with Waardenburg syndrome type 1（WS1）, and the mother of patient 1, patient 2, patient 3, and the mother of patient 3 with WS2. Conclusion:WS is a syndromic deafness, and the main clinical features include autosomal dominant inheritance and scleral pigment abnormalities. However, the findings of this study show that there is still phenotypic heterogeneity in WS even caused by the same gene variant, so it depends on genetic tests to confirm the diagnosis; The gene variant of patient 1 and 2 was never been reported in other patients, which expands the pathogenic variant spectrum of WS.

Long intergenic non-protein coding RNA 02570 promotes nasopharyngeal carcinoma progression by adsorbing microRNA miR-4649-3p thereby upregulating both sterol regulatory element binding protein 1, and fatty acid synthase.

Our previous studies have elucidated a possible connection between long intergenic non-protein coding RNA 2570 (LINC02570) and nasopharyngeal carcinoma (NPC). However, the precise mechanism by which LINC02570 promotes NPC remains unknown. We used quantitative polymerase chain reaction (qPCR) to detect LINC02570 expression in nasopharyngeal cell lines, NPC tissues, and chronic rhinitis tissues. Subcellular LINC02570 localization was confirmed by fluorescence in situ hybridization (FISH). The effects of LINC02570 stable knockdown and overexpression on viabillity, proliferation, migration, and invasion were analyzed using 3-(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl-2-H-Tetrazolium bromide (MTT), a colorimetric focus-formation assay, a wound healing assay, and transwell assays. RNA crosstalk analysis in silico predicted microRNA-4649-3p (miR-4649-3p) binding to LINC02570 or sterol regulatory element binding transcription factor 1 (SREBF1). A dual luciferase reporter assay was used to confirm potential interactions. Sterol regulatory element binding protein 1 (SREBP1) and fatty acid synthase (FASN) expression were detected by western blotting. The results suggest that LINC02570 is upregulated in late clinical stage NPC patients, and promotes NPC progression by adsorbing miR-4649-3p to up-regulate SREBP1 and FASN. This study elucidates a potential chemotherapeutic target involved in lipid metabolism in NPC.

[Identification of novel pathogenic variants of TRIOBP gene in a pedigree affected with non-syndromic deafness].

OBJECTIVE: To explore the genetic basis for a Chinese pedigree affected with non-syndromic hearing loss (NSHL). METHODS: Commercialized gene chip was applied to detect common mutations associated with congenital deafness. Whole exome sequencing was carried out for patients for whom gene chip yielded a negative result. Candidate variants were verified by Sanger sequencing. RESULTS: Two patients from the pedigree were discovered to carry compound heterozygous variants of the TRIOBP gene, namely c.3299C>A and c.5185-2A>G. Their parents had normal hearing and were both heterozygous carriers of the above variants. Both variants had co-segregated with the disease phenotype in the pedigree and were unreported previously. CONCLUSION: Pathogenic variants of the TRIOBP gene comprise an important factor for NSHL. The novel c.5185-2A>G and c.3299C>A variants discovered in this study have enriched the mutational spectrum of the TRIOBP gene and enabled molecular diagnosis and genetic counseling for the family.

A novel SLC26A4 splicing mutation identified in two deaf Chinese twin sisters with enlarged vestibular aqueducts.

BACKGROUND: Variants in the SLC26A4 gene are correlated with nonsyndromic hearing loss with an enlarged vestibular aqueduct (EVA). This study aimed to identify the genetic causes in a Chinese family with EVA, and the pathogenicity of the detected variants. METHODS: We collected blood samples and clinical data from a pair of deaf twin sisters with EVA and their family members. As controls, a group of 500 normal-hearing people were enrolled in our study. Twenty-one exons and flanking splice sites of the SLC26A4 gene were screened for pathogenic mutations by polymerase chain reaction and bidirectional Sanger sequencing. Minigene assays were used to verify whether the novel SLC26A4 intronic mutation influenced the normal splicing of mRNA. RESULTS: Hearing loss in the twins with EVA was diagnosed using auditory tests and imaging examinations. Two pathogenic mutations, c.919-2A>G and c.1614+5G>A were detected in SLC26A4, the latter of which has not been reported in the literature. The minigene expression in vitro confirmed that c.1614+5G>A could cause aberrant splicing, resulting in skipping over exon 14. CONCLUSIONS: On the SLC26A4 gene, c.1614+5G>A is a pathogenic mutation. This finding enriches the mutational spectrum of the SLC26A4 gene and provides a basis for the genetic diagnosis of EVA.

[Current status and new progress of vestibular assistant systems].

Vestibular dysfunction can seriously affect patients' daily life and work. There is an urgent need for new methods to treat vestibular dysfunction. As awareness of these diseases increases, efforts are being made to improve vestibular function through a number of vestibular assistant systems. As far as current development is concerned, vestibular assistant system mainly focuses on two fields: external prosthesis and vestibular implant. This review shows the current status and future development trend of the vestibular assistant system.

[Study on Dynamic Changes of Human Papillomavirus 16 E5 Gene in the Occurrence of Cervical Cancer].

OBJECTIVE: To explore the dynamic changes of human papillomavirus (HPV) type 16 E5 gene in the development of cervical cancer and the significance of E5 mRNA in early screening of cervical cancer. METHODS: Paraffin specimens of cervical lesions were collected from 49 cases (HPV positive) during September 2015 to December 2017 According to the standard of FIGO, all cervical lesions were diagnosed as: 13 cases of cervicitis, cervical intraepithelial neoplasia disorders (CIN) Ⅰ in 5 cases, CIN Ⅱ in 18 cases, CIN Ⅲ in 5 cases, 8 cases of cervical cancer. Real-time fluorescence quantitative PCR was used to detect the integrity of E5 gene and the mRNA expression levels of E5, E6 and E 7in cervical tissues. RESULTS: All the 49 cases showed positive HPV16 infection. E5 genetic integrity in CINⅠwas higher than that in cervical inflammation, CIN Ⅱand cervical cancer (P < 0.05), which was also higher than that in CIN Ⅲ, but without statistically significance (P>0.05). The mRNA levels of E5, E6, E7 were the highest in CIN Ⅲ. Compared with E6 and E7, E5 presented superior expression in all types of cervical lesions (P < 0.05), while E 6and E7 mRNA expressions only increased in CIN Ⅲ and cervical cancer. CONCLUSION: In the patients with HPV16 infection, the integrity of E5 gene in cervical tissues may be related to the occurrence and development of cervical diseases. E5 gene is expected to be the target gene for early screening of cervical cancer.

Long Noncoding RNAs and Messenger RNAs Expression Profiles Potentially Regulated by ZBTB7A in Nasopharyngeal Carcinoma.

Our previous studies showed that ZBTB7A played an important role in promoting nasopharyngeal carcinoma (NPC) progression. However, molecular mechanisms of different levels of ZBTB7A are still unclear. It is necessary to search molecular markers which are closely connected with ZBTB7A. We selected NPC sublines CNE2 with stably transfecting empty plasmid (negative control, NC) and short hair RNA (shRNA) plasmid targeting ZBTB7A as research objectives. Microarray was used to screen differentially expressed long noncoding RNAs (lncRNAs) and messenger RNAs (mRNAs) via shRNA-CNE2 versus NC-CNE2. Quantitative PCR (qPCR) was used to validate lncRNAs and mRNAs from the sublines, chronic rhinitis, and NPC tissues. Bioinformatics was used to analyze regulatory pathways which were connected with ZBTB7A. The 1501 lncRNAs (long noncoding RNAs) and 1275 differentially expressed mRNAs were upregulated or downregulated over 2-fold. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the upregulated or downregulated carbohydrate and lipid metabolisms probably involved in carcinogenicity of shRNA-CNE2 (P-value cut-off was 0.05). In order to find the molecular mechanisms of ZBTB7A, we validated 12 differentially expressed lncRNAs and their nearby mRNAs by qPCR. Most of the differentially expressed mRNAs are closely connected with carbohydrate and lipid metabolisms in multiply cancers. Furthermore, part of them were validated in NPC and rhinitis tissues by qPCR. As a result, NR_047538, ENST00000442852, and fatty acid synthase (FASN) were closely associated with NPC. ZBTB7A had a positive association with NR_047538 and negative associations with ENST00000442852 and FASN. The results probably provide novel candidate biomarkers for NPC progression with different levels of ZBTB7A.

Stable knockdown of ZBTB7A promotes cell proliferation and progression in nasopharyngeal carcinoma.

AIMS: Although high expression of ZBTB7A is positively relative to metastasis in nasopharyngeal carcinoma (NPC) patients, the association between its low expression and metastasis of NPC remains unclear. The present study aimed to definitely identify the association. METHODS: The level of ZBTB7A was effectively knocked down by stable transfection of short hair RNA plasmid in NPC cell lines CNE2 and 5-8F (shRNA-CNE2 and shRNA-5-8F), compared with the cells that stably transfected empty plasmid (NC-CNE2 and NC-5-8F). The levels of ZBTB7A were assessed by real-time polymerase chain reaction and Western blot in the cell lines. MTT assay, colorimetric focus-formation assay, flow cytometry, wound healing assay, transwell assays, and xenograft model were performed to analyze cell vitality, proliferation, cell cycle, migration, invasion, and tumorigenicity. RESULTS: The levels of ZBTB7A were effectively reduced in shRNA-CNE2 and shRNA-5-8F. Their carcinogenicity was stronger separately than the abilities of NC-CNE2 and NC-5-8F. NC-CNE2 and shRNA-CNE2 were selected to establish the xenograft model because of their stronger tumorigenicity than NC-6-10B and shRNA-5-8F. The assay showed that shRNA-CNE2 had stronger tumorigenicity than NC-CNE2. CONCLUSIONS: The results demonstrated the reverse association between the expression of ZBTB7A and the tumorigenicity of NPC. We postulate that some oncogenic pathways, which are suppressed by ZBTB7A, will vicariously promote the proliferation and progression of NPC when ZBTB7A is decreased.

Novel compound heterozygous mutations in the OTOF Gene identified by whole-exome sequencing in auditory neuropathy spectrum disorder.

BACKGROUND: Many hearing-loss diseases are demonstrated to have Mendelian inheritance caused by mutations in single gene. However, many deaf individuals have diseases that remain genetically unexplained. Auditory neuropathy is a sensorineural deafness in which sounds are able to be transferred into the inner ear normally but the transmission of the signals from inner ear to auditory nerve and brain is injured, also known as auditory neuropathy spectrum disorder (ANSD). The pathogenic mutations of the genes responsible for the Chinese ANSD population remain poorly understood. METHODS: A total of 127 patients with non-syndromic hearing loss (NSHL) were enrolled in Guangxi Zhuang Autonomous Region. A hereditary deafness gene mutation screening was performed to identify the mutation sites in four deafness-related genes (GJB2, GJB3, 12S rRNA, and SLC26A4). In addition, whole-exome sequencing (WES) was applied to explore unappreciated mutation sites in the cases with the singularity of its phenotype. RESULTS: Well-characterized mutations were found in only 8.7% (11/127) of the patients. Interestingly, two mutations in the OTOF gene were identified in two affected siblings with ANSD from a Chinese family, including one nonsense mutation c.1273C > T (p.R425X) and one missense mutation c.4994 T > C (p.L1665P). Furthermore, we employed Sanger sequencing to confirm the mutations in each subject. Two compound heterozygous mutations in the OTOF gene were observed in the two affected siblings, whereas the two parents and unaffected sister were heterozygous carriers of c.1273C > T (father and sister) and c.4994 T > C (mother). The nonsense mutation p.R425X, contributes to a premature stop codon, may result in a truncated polypeptide, which strongly suggests its pathogenicity for ANSD. The missense mutation p.L1665P results in a single amino acid substitution in a highly conserved region. CONCLUSIONS: Two mutations in the OTOF gene in the Chinese deaf population were recognized for the first time. These findings not only extend the OTOF gene mutation spectrum for ANSD but also indicate that whole-exome sequencing is an effective approach to clarify the genetic characteristics in non-syndromic ANSD patients.

[Analysis common gene mutation spots of 127 non-syndromic deafness natients in Guangxi Drovince].

OBJECTIVE: To investigate the mutation characteristics of common deafness gene from 127 non-syndromic hearing loss patients in Guangxi province. METHOD: Deafness-related gene mutations detection kit was used to detect 15 mutation sites in four deafness-associated genes, and a total of 127 hearing impaired patients were tested. The samples that could not be diagnosed with DNA microarray were subjected to PCR and sequenced to detect other mutations. RESULT: Among the 127 patients with non-syndromic deafness, the total mutation rate is 8.66% (11/127), including GJB2 235delC homozygous in 3 cases (2.36%), 235delC single heterozygous mutation in 2 cases (1.57%), GJB2 235delC and 109 A > G mutations in 2 cases (1.57%); SLC26A4 1229C > T homozygous in 1 case (0.79%), IVS7-2A > G, IVS11 + 47T > C and 15448insC mutaion in 2 cases (1.57%); mitochondrial 12S rRNA gene mutations were not detected. The result indicates that GJB2 and SLC26A4 were the main genes in this study, and the mutation rate is significantly lower than the national average level. Three new mutations (SLC26A4 IVS11 + 47T > C,1548insC and GJB2 109A > G) were found. There may be rare mutations among sites or genes associated with deafness in Guangxi.

[Diagnosis and management of jugulare glomus tumor and carotid body tumor].

OBJECTIVE: To investigate diagnosis method and treatment experience of jugulare glomus tumor (JGT) and carotid body tumor (CBT). METHOD: Retrospective analysis. Clinical materials from 4 patients at one genealogy with JGT and CBT were collected. The clinical features, radiological characteristics, surgical methods and prognosis were assessed. The proband suffered from left JGT and left CBT, her younger sister had right CBT as well as her younger female cousin had bilateral CBT and right JGT, her older male cousin had bilateral CBT and left JGT. These JGT and CBT in four patients were resected under general anesthesia after preoperative ultrasound, CT or CT angiography (CTA), MRI, and digital subtraction angiography (DSA) examination as well as preoperative vascular embolization. RESULT: The surgery was uneventful in four cases, and there was no hemiplegia or deaths. All patients were followed up for 2 years to 29 months without recurrence. Six months after operation, the hoarseness also disappeared in the older male cousins but did not improve significantly in younger female cousin. The younger female cousin get right peripheral facial paralysis 1 year after surgery and her temporal bone CT indicated a right JGT. CONCLUSION: It is very important to assess JGT and CBT by Ultrasound, CT, CTA, MRI and DSA. Surgical resection is the first optional treatment for JGT and CBT. Blood loss can be reduced by preop erative vascular embolization. Serious complications could be avoid by operating under microscope, which can supply a clear surgical field and make the surgeon to protect the large blood vessels and nerve.

Expression of the Pokemon proto-oncogene in nasopharyngeal carcinoma cell lines and tissues.

To study the differentiated expression of the proto-oncogene Pokemon in nasopharyngeal carcinoma (NPC) cell lines and tissues, mRNA and protein expression levels of CNE1, CNE2, CNE3 and C666-1 were detected separately by reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and Western-blotting. The immortalized nasopharyngeal epithelial cell line NP69 was used as a control. The Pokemon protein expression level in biopsy specimens from chronic rhinitis patients and undifferentiated non keratinizing NPC patients was determined by Western-blotting and arranged from high to low: C666-1>CNE1>CNE2> CNE3>NP69. The Pokemon mRNA expression level was also arranged from high to low: CNE1>CNE2>NP69>C666-1>CNE3. Pokemon expression of NP69 and C666-1 obviously varied from mRNA to protein. The Pokemon protein level of NPC biopsy specimens was obviously higher than in chronic rhinitis. The data suggest that high Pokemon protein expression is closely associated with undifferentiated non-keratinizing NPC and may provide useful information for NPC molecular target therapy.

[Study for the histopathologic change of ethmoid bone in patients with chronic rhinosinusitis and its correlation factors].

OBJECTIVE: To investigate the histopathologic changes of ethmoid bone and its correlation with clinical types of chronic rhinosinusitis (CRS). METHOD: All ethmoid bones and mucosa from 180 patients with CRS after endoscopic sinus surgery were collected for histopathologic detection with HE staining. The number and the rate of cases were counted according to different histopathologic types. To analyze the correlation between ethmoid bones and clinical types of CRS, mucosal pathologic change, the CT-scanning types of sinusitis, the course of disease as well as operational history. RESULT: The ethmoid bone of all patients had varying degrees of histopathologic changes. There were 5 cases (2.78%) in stage I, 38 cases (21.11%) in stage II, 71 cases (39.44%) in stage III, and 66 cases (36.67%) in stage NIV. The histopathologic changes of ethmoid bone varied in different clinical types. In type I, there were 5 cases (8.33%) in stage I, 33 cases (55.00%) in stage II, 15 cases (25.00%) in stage III, and 7 cases (11.67%) in stage NV. In type I, there were 5 cases (8.33%) in stage II, 37 cases (61.67%) in stage mI, and 18 cases (30.00%) in stage NV. In type III, there were 19 cases (31.67%) in stage III, and 41 cases (68.33%) in stage NV. All histopathologic changes of ethmoid bone were statistically correlated (P < 0.01) with clinical types of CRS, pathologic mucosal change, the CT-scanning types of sinusitis, the course of disease as well as operational history. CONCLUSION: Almost all patients with CRS manifest different-degrees of histopathologic changes, which are correlated with the clinical types of CRS, pathologic mucosal change, the CT-scanning types, the course of disease as well as operational history.

[Clinical study on modified transpalatine radical operation for carcinoma of nasopharynx].

OBJECTIVE: To evaluate the method of transpalatine surgical treatment of carcinoma of nasopharynx (NPC). METHOD: The artery palatine major was protected and the resection domain of primary lesion of NPC was expanded by modified hard palatine incision. Preventive radiotherapy and routine chemotherapy was given after the operation. RESULT: Twelve patients were in our report, five of II stage and seven of III stage. In all cases, the incisions in hard palatine were healing by first intention after the operations. The patients were followed up for six months to five years. None case was found relapse or palatal fistula. They all can swallow and speech as normal. Sequelae of radiotherapy was not found. CONCLUSION: Without affecting the healing of the incision, expanding the resective domain of primary lesion of NPC is possible by modified hard palatine incision, and the occurrence of sequelae of radiotherapy can be reduced by giving preventive radiotherapy.

[Clinical study of non-invasive nasal sinus mycoses].

OBJECTIVE: To explore the pathogeny, diagnosis and treatment of the noninvasive nasal sinus mycoses. METHOD: 11 cases with nasal sinus mycoses were analyzed retrospectively. All of them had undergone endoscopic operation. RESULT: After operation all cases got regular check up under endoscope. 9 cases recovered quickly. The other 2 had caseous matter in their maxillary sinus in the initial stage. After cleaning up the fungus ball and washing the maxillary sinus for several times, the surgical cavity became clear. 10 cases had been followed up for 6 months to 5 years and no recurrence occurred. The other one lost follow-up. CONCLUSION: Endoscopic operation is the basic surgical method to treat the non-invasive nasal sinus mycoses. Excising the focus thoroughly, correcting the abnormal nasal cavity structure and resuming the cleanout function of nasal sinus cilia are the principle of treatment.